Author:
Bkaily G,Sleiman S,Stephan J,Asselin C,Choufani S,Kamal M,Jacques D,Gobeil, Jr F,D'Orléans-Juste P
Abstract
The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R GFP overexpressing hVSMCs, Ang II (109 and 104 M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1RGFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1RGFP overexpressing hVSMCs, Ang II (1015 to 104 M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 × 1011 and 5 × 109 M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.Key words: angiotensin, calcium, protein synthesis, nucleus, AT1 receptor, nuclear de novo synthesis.
Publisher
Canadian Science Publishing
Subject
Physiology (medical),Pharmacology,General Medicine,Physiology
Cited by
67 articles.
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